Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/ peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.The outer segments (OSs) 1 of rod and cone photoreceptor cells act as detectors of visible light for the initial stages of the visual process and are vital for normal vertebrate vision. The OS is an architecturally complex organelle. It contains hundreds of precisely stacked membranous disks that undergo a polarized renewal process; a complete turnover of OS membrane protein occurs approximately once every 10 days in primate rod cells (1). Although the renewal process and OS stability are essential for photoreceptor viability, their underlying molecular bases remain largely undefined. Evidence from several laboratories has implicated the integral membrane protein peripherin/rds in OS morphogenesis and the renewal process (2-4). It is present in all vertebrate rod and cone photoreceptors examined to date and causes a variety of progressive retinal diseases in humans when defective (5).Despite continued interest ...
The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(؉)--hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.Rhodobacter sphaeroides has a diverse array of catabolic pathways at its command, including aerobic and anaerobic respiration and photosynthesis (for reviews, see references 24 and 51). These metabolisms are supported by the ability of R. sphaeroides to synthesize several metallotetrapyrroles, including hemes, bacteriochlorophyll (bchl), and corrinoids (reviewed in reference 23). Organisms commanding such metabolic versatility must have regulatory mechanisms that confer the ability to choose among these pathways in order to maximize energy production with the resources available and according to the prevailing environmental conditions. A readily observable indication of the presence of these mechanisms is the variation in absolute and relative levels of the different tetrapyrroles present in R. sphaeroides according to the catabolic state of the cell. The level of bchl undergoes a dramatic increase under conditions of lowering oxygen tension in preparation for anoxygenic photosynthetic energy production (29). On the other hand, heme levels must be maintained for aerobic and anaerobic respiration, as well as for photosynthesis (reviewed in references 23 and 29). The corrinoid vitamin B 12 is an essential cofactor in methionine synthesis in this organism (10; for a general review, see reference 43) and thus is required under all conditions. One control point for regulated production of tetrapyrroles is at the level of formation of their common precursor, 5-aminolevulinic acid (ALA).It is well established in R. sphaeroides that the levels of ALA formation parallel the amount of tetrapyrroles in the cell (29). However, the presence of more than one gene coding for ALA synthase activity in R. sphaeroides 2.4.1 (37, 48) requires knowledge of the expression of each gene in order to achieve a full understanding of how ALA levels are regulated. R. sphaeroides is the only known prokaryote possessing duplicate genes for ALA synthase, and the genes coding for the two isozymes, hemA and hemT, are diff...
The presumed DNA target for the Rhodobacter sphaeroides 2.4.1 FnrL regulatory protein is the FNR consensus sequence, TTGAT-N(4)-ATCAA, based on (1) similarities between the helix-turn-helix motifs of FnrL and the Escherichia coli homologue, Fnr, and (2) the established FnrL dependence for anaerobic induction of six gene clusters all having upstream FNR consensus-like sequences. We are interested in understanding the regulation of one among these; namely, the hemA gene, which codes for one of two isozymes of 5-aminolevulinate (ALA) synthase in this organism. Here, we present in vivo evidence that the hemA gene is transcribed from two promoters. Both are under oxygen control, and disabling the fnrL gene abolishes induction of each promoter in response to lowering oxygen tension. Based on the 5' position of the FNR consensus sequence relative to the downstream promoter, we had hypothesized that activation of that promoter is mediated by binding of FnrL to the FNR consensus sequence. Consistent with this hypothesis, we found here that transcription from the downstream promoter is no longer inducible when the FNR consensus sequence is deleted. With respect to the upstream promoter, based on the fact that the +1 site of transcription from that promoter is within the FNR consensus sequence, we propose an indirect role for FnrL. This possibility is discussed, together with other unresolved aspects of hemA expression.
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